New uses for old drugs: the tale of artemisinin derivatives in the elimination of schistosomiasis japonica in China.

Artemisinin (qinghaosu), extracted from the Chinese herb Artemisia annua L. in 1972, and its three major derivatives--artemether, artesunate and dihydroartemisinin--were firstly identified as antimalarials and found active against all species of the malaria parasite. Since the early 1980s, artemisinin and its derivatives have been found efficacious against Schistosoma spp., notably larval parasites, and artemisinin derivatives have played a critical role in the prevention and treatment of human schistosomiasis in China. Currently, China is moving towards the progress of schistosomiasis elimination. However, the potential development of praziquantel resistance may pose a great threat to the progress of elimination of schistosomiasis japonica in China. Fortunately, these three major artemisinin derivatives also exhibit actions against adult parasites, and reduced sensitivity to artemether, artesunate and dihydroartemisinin has been detected in praziquantel-resistant S. japonicum. In this review, we describe the application of artemisinin derivatives in the prevention and treatment of schistosomiasis japonica in China, so as to provide tools for the global agenda of schistosomiasis elimination. In addition to antimalarial and antischistosomal actions, they also show activities against other parasites and multiple cancers. Artemisinin derivatives, as old drugs identified firstly as antimalarials, continue to create new stories.

Gene profile of chemokines on hepatic stellate cells of schistosome-infected mice and antifibrotic roles of CXCL9/10 on liver non-parenchymal cells.

Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis caused by schistosomiasis. Chemokines were widely expressed and involved in cellular activation, proliferation and migration in inflammatory and infectious diseases. However, little is known about the expressions of chemokines on HSCs in the schistosoma infection. In addition, the roles of chemokines in pathogenesis of liver fibrosis are not totally clear. In our study, we used microarray to analyze the temporal gene expressions of primary HSCs isolated from mice with both acute and chronic schistosomiasis. Our microarray data showed that most of the chemokines expressed on HSCs were upregulated at 3 weeks post-infection (p.i) when the egg granulomatous response was not obviously evoked in the liver. However, some of them like CXCL9, CXCL10 and CXCL11 were subsequently decreased at 6 weeks p.i when the granulomatous response reached the peak. In the chronic stage, most of the differentially expressed chemokines maintained persistent high-abundances. Furthermore, several chemokines including CCR2, CCR5, CCR7, CXCR3, CXCR4, CCL2, CCL5, CCL21, CXCL9 and CXCL10 were expressed by HCSs and the abundances of them were changed following the praziquantel treatment in the chronic stage, indicating that chemokines were possibly necessary for the persistence of the chronic stage. In vitro experiments, hepatic non-parenchymal cells, primary HSCs and human HSCs line LX-2 were stimulated by chemokines. The results showed that CXCL9 and CXCL10, but not CXCL11 or CXCL4, significantly inhibited the gene expressions of Col1α1, Col3α1 and α-SMA, indicating the potential anti-fibrosis effect of CXCL9 and CXCL10 in schistosomiasis. More interestingly, soluble egg antigen (SEA) of Schistosoma japonicum was able to inhibit transcriptional expressions of some chemokines by LX-2 cells, suggesting that SEA was capable of regulating the expression pattern of chemokine family and modulating the hepatic immune microenvironment in schistosomiasis.

Murine CD8(+)T cell cytotoxicity against schistosomula induced by inoculation of schistosomal 22.6/26GST coupled Sepharose 4B beads.

Schistosomasis is a world-wide parasitic disease. Although chemotherapy is the main treatment method for schistosomasis currently, it cannot prevent schistosome reinfection. Up to now no effective vaccine is available to prevent schistosomiasis. Dendritic cells (DCs) are one of the key players in the cellular immune response and play an important role in antigen presentation as antigen-presenting cells. Here we reported a novel large particulate antigen, in which Sepharose 4B beads were coated with Sj22.6/26GST. Our results showed that this particulate antigen could be cross-presented by DCs to CD8(+)T cells. Furthermore, CD8(+)T cells stimulated by particulate antigen directly exerted cytotoxicity against Schistosoma japonicum schistosomula. We also demonstrated that S. japonicum schistosomula acquired the MHC class I molecules from host blood serum and presented the molecules at the larval surface. While it may help them escape from the host immune surveillance, these MHC I-antigen complexes presented on the surface render schistosomula the potential targets of the CD8(+)T cell cytotoxicity induced by particulate antigen-based vaccine. Finally we evaluated the protective immunity of this particulate vaccine in a mouse infection challenge model. Our data clearly showed that the particulate vaccine induced a partial reduction in both worm burdens and egg loads. Taken together, these results suggest that this large particulate vaccine could be a potential vaccine for the prevention of schistosome infection.

[Effects of excreted/secreted antigens of Toxoplasma gondii on CD4+ CD25+ Foxp3+ T cells and NK cells of melanoma-bearing mice].

OBJECTIVE: To explore the effects of excreted/secreted antigens (ESA) of Toxoplasma gondii on CD4+CD25+ Foxp3+ T cells and NK cells of melanoma-bearing mice, as well as the tumor growth. METHODS: B16F10 (denoted B16) tumor cells were cultured in complete medium and maintained by serial passage in vitro. The 2x 10(5) B16 tumor cells were injected into the right flank of the mouse to establish the tumor-bearing mice model. Mice were randomly divided into four groups, namely PBS, B16F10, PES/ESA and B16F10/ESA groups after ESA injections. On days 2, 4 and 6 post-ESA injection, the spleens were removed. The percentage of CD4+CD25+ Foxp3+ T cells and NK cells in splenocytes were determined by flow cytometry; the suppression functions of CD4+CD25+ Tregs and the NK cell activity were detected by WST-8 and LDH methods, respectively. The tumor growth of each group was measured. RESULTS: On Days 4 and 6 post-ESA injection, the percentages of CD4+CD25+ Foxp3+ T cells in splenocytes of the B16F10/ESA-injected mice decreased being (1.65 +/- 0.18)% and (1.56 +/- 0.17)%, respectively, and compared with those in the B16-injected mice [(2.47 +/- 0.10)% and (2.82 +/- 0.12)%], there were significant differences (both P values < 0.05). The inhibition of CD4+CD25+ Tregs of the B16F10/ESA-injected mice decreased markedly on Day 4 (50.03%) and Day 6 (50%) compared with those in the control (75.03% and 78.14%) post-ESA injection, there were significant difference (both P values < 0.05). The percentages of NK cells in splenocytes on Day 6 post-ESA injection [(3.58 +/- 0.07)%] was significantly higher than that of control [(2.61 +/- 0.13)%]. The activities of NK cells from B16F10/ESA - injected mice against B16 cells at different effect - to - target cell ratios (5 : 1, 10 :1, 20 : 1), increased significantly being 26.51%, 35.25%, 60.19%, respectively, while compared with those in the control (16.81%, 24.63% and 45.62%), there were significant differences (all P values < 0.05). In addition, the volume of the B16 tumors [(6 208.34 +/- 443.64)]mm3 was significantly smaller than that of control [(9 027.46 +/- 1 362.01)] mm3(P < 0.05) when measured at Day 35 post-tumor innoculation. CONCLUSIONS: T. gondii ESA can downregulate CD4+CD25+Tregs while upregulating NK cells of B16 tumor-bearing mice quantitatively and functionally, therefore plays a role in suppression of tumor growth.

[Anti-hepatofibrosis effect of fasudil hydrochloride on Schistosoma japonicum-infected mice].

OBJECTIVE: To investigate the anti-fibrotic effect of fasudil hydrochloride on Schistosoma japonicum-infected mice, and the effect of fasudil hydrochloride on hepatic stellate cells (HSCs). METHODS: Thirty female BALB/c mice were randomly divided into 3 groups viz, normal control group (NC group), infection group, and experiment group. Mice in both infection group and experiment group were infected with (14:2) cercariae of S japonicum. At 6 weeks post infection, mice in experiment group were intraperitoneally injected with fasudil hydrochloride (10 mg/kg) twice a day for 7 d, while mice in NC group and infection group received the same volume of physiological saline. All mice were sacrificed 12 h after the last injection. Livers from NC group and infection group were used to prepare tissue sections for hematoxylin and eosin (HE) staining, or sirius red staining, and observed under light microscope. Livers from all three groups were used to detect content of hydroxyproline (Hyp) and the mRNA expressions of alpha-smooth muscle actin (alpha-SMA), type I collagen alpha1 (Col1alpha1) and epithelial cell transforming sequence 2 (Ect2). HSCs from mice in all three groups were isolated to detect the mRNA levels of alpha-SMA, Col1alpha1, and Ect2, respectively. RESULTS: Pathological sections showed that in livers from mice in infection group, inflammatory cells infiltrated and collagenous fibre proliferated around portal areas and egg granulomas. The content of Hyp in liver from mice of NC group, infection group, and experiment group was (279.7 +/- 21.2) microg, (528.0 +/- 15.0) microg, and (355.4 +/- 22.6) microg, respectively. The content of Hyp in livers from mice of experiment group was significantly reduced compared to infection group (P < 0.01). The mRNA expression of alpha-SMA, Col1alpha1 and Ect2 in livers and HSCs from mice in experiment group were significantly down-regulated compared to infection group (P < 0.05). CONCLUSION: Fasudil hydrochloride can depress hepatofibrosis in Schistosoma japonicum-infected mice.

New insight into the antifibrotic effects of praziquantel on mice in infection with Schistosoma japonicum.

BACKGROUND: Schistosomiasis is a parasitic disease infecting more than 200 million people in the world. Although chemotherapy targeting on killing schistosomes is one of the main strategies in the disease control, there are few effective ways of dealing with liver fibrosis caused by the parasite infection in the chronic and advanced stages of schistosomiasis. For this reason, new strategies and prospective drugs, which exert antifibrotic effects, are urgently required. METHODS AND FINDINGS: The antifibrotic effects of praziquantel were assessed in the murine models of schistosomiasis japonica. Murine fibrosis models were established by cutaneous infection with 14 ± 2 Schistosoma japonicum cercariae. Then, the mice of both chronic (8 weeks post-infection) and advanced (15 weeks post-infection) schistosomiasis were treated by gavage of praziquantel (250 mg/kg, once daily for 3 days) to eliminate worms, and followed by praziquantel anti-fibrosis treatment (300 mg/kg, twice daily for 30 days). The fibrosis-related parameters assessed were areas of collagen deposition, content of hydroxyproline and mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-ß, MMP9, TIMP1, IL-4, IL-10, IL-13 and IFN-γ of liver. Spleen weight index, alanine aminotransferase activity and liver portal venous pressure were also measured. The results showed that anti-fibrosis treatment improved liver fibrosis, splenomegaly, hepatic function, as well as liver portal hypertension. In order to confirm the anti-fibrotic properties of praziquantel, we established a CCL(4)-induced model and revealed that CCL(4)-induced liver fibrosis was inhibited by PZQ treatment for 30 days. Furthermore, we analyzed the effects of praziquantel on mouse primary hepatic stellate cells (HSCs). It is indicated that mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-ß, MMP9 and TIMP1 of HSCs were all inhibited after praziquantel anti-parasite treatments. CONCLUSIONS: The significant amelioration of hepatic fibrosis by praziquantel treatment validates it as a promising drug of anti-fibrosis and offers potential of a new chemotherapy for hepatic fibrosis resulting from schistosomiasis.

[Effect of Schistosoma japonicum Mr 22 600 particulated-antigen on dendritic cells and CD4+CD25+ regulatory T cells].

OBJECTIVE: To study the role of Schistosoma japonicum Mr 22 600 particulated-antigen on dendritic cells (DCs) and CD4+CD25+ regulatory T cells. METHODS: In in vitro experiments, DCs were pulsed with Sepharose 4B coupling rSj22.6/26GST and soluble rSj22.6/26GST, respectively. The surface molecules of DCs were detected by flow cytometry and the function of DCs was detected by mixed lymphocyte reaction. For the reduction of CD4+CD25+ T cells, DCs pulsed with Sepharose 4B coupling rSj22.6/26GST and soluble rSj22.6/26GST, respectively, were co-cultured with CD4+ T cells isolated from the spleen cells. The percentage of CD4+CD25+ Foxp3+ T cells in CD4+ T cells was detected by flow cytometry. In in vivo experiments, BALB/c mice were immunized with Sepharose 4B coupling rSj22.6/26GST, Freund's adjuvant emulsified rSj22.6/26GST, rSj22.6/26GST, Sepharose 4B, Freund's adjuvant and PBS, respectively. The percentage of DCs in draining lymph nodes and the percentage of CD4+CD25+Foxp3+ T cells in spleen cells were detected by flow cytometry. To analyze the inhibitory roles of CD4+CD25+ T cells on CD4+CD25- T cells, CD4+CD25+ T cells were separated from mice immunized with Sepharose 4B coupling rSj22.6/26GST and Freund's adjuvant emulsified rSj22.6/26GST, respectively, and co-cultured with CD4+CD25- T cells. The proliferation of cells was assessed by [3H] thymidine incorporation method. RESULTS: In vitro, the expression rate of the surface molecules of CD40, CD80 and CD86 on the soluble antigen pulsed DCs were (43.5 +/- 6.2)%, (37.7 +/- 0.1)%, and (71.4 +/- 1.4)%, respectively. But on the Sepharose 4B coupling antigen pulsed DCs, they were (31.2 +/- 5.4)%, (32.0 +/- 1.6)%, and (63.8 +/- 1.0)%, respectively, which suggested that the Sepharose 4B coupling rSj22.6/26GST had less stimulating roles on DCs maturation Furthermore, addition of DCs pulsed with Sepharose 4B coupling rSj22.6/26GST caused the expanding of CD4+CD25+ T cells. In vivo, immunization of Sepharose 4B coupling rSj22.6/26GST increased the number of CD4+CD25+ T cells. CD4+CD25+ T cells separated from Sepharose 4B coupling rSj22.6/26GST immunized mice had stronger inhibitory ability (cpm 1 420 +/- 335), compared with that of mice immunized with soluble antigen (cpm 3 558 +/- 147). CONCLUSION: In contrast to the Freund's adjuvant emulsified antigen, immunization with Sepharose 4B coupling rSj22.6/26GST increases the number of CD4+CD25+ T cells, which showed stronger inhibition on the CD4+ CD25- T cell proliferation, and the mechanism of which may be involved in DCs maturation.

[Suppressive effect of induced CD4+CD25+ regulatory T cells from mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the suppressive effect of CD4+CD25+ regulatory T cells from mice infected with Schistosoma japonicum. METHODS: BALB/c mice were infected with S. japonicum. At 6 and 13 weeks post-infection, the spleens were removed and CD4+CD25+ T cells were separated by magnetic beads. In in vitro experiments, CD4+CD25+ T Cells were cocultured with CD4+CD25- T cells. The inhibitory role of the CD4+CD25+ T cells was assessed by [3H] thymidine incorporation method and the cytokines in the cultural supernatant were detected by ELISA. In in vivo experiments, mice inoculated with irradiated cercariae of S. japonicum were adoptively transferred with CD4+CD25+ T cells isolated from the mice chronically infected with S. japonicum. The intracellular cytokine expressions of splenocytes were performed by flow cytometry, and sera IgG1 and IgG2a antibodies against irradiated cercaria antigens were detected by ELISA. RESULTS: In vitro, CD4+CD25+ T cells were able to suppress the proliferation of CD4+CD25- T cells when stimulated with SEA, compared with single CD4+CD25- T cells culture (cpm 7615 +/- 1 058) (P < 0.01). Furthermore, CD4+CD25+ T cells isolated from mice chronically infected with S. japonicum presented higher suppressive efficacy (cpm 2 336 +/- 490), compared with that isolated from the acutely infected mice (cmp 4 467 +/- 144) (P < 0.05). Meanwhile, CD4+CD25+ T cells isolated from mice with the acute infection inhibited the cytokine secretion by CD4+CD25- T cells and the suppression rate was 32.0% for IL-4 (P < 0.05), 66.3% for IFN-gamma (P < 0.01) and 63.2% for IL-2 (P < 0.01), respectively, and CD4+CD25+ T cells isolated from mice with the chronic infection, the suppression rate was 28.4% for IL-4 (P < 0.05), 60.1% for IFN-gamma (P < 0.01) and 58.3% for IL-2 (P < 0.01), respectively. In vivo, IFN-gamma secretion and IgG2a antibody production of mice adoptively transferred with CD4+CD25+ T cells from the chronically infected mice were suppressed when mice were inoculated with irradiated cercariae of S. japonicum (P < 0.05). CONCLUSION: CD4+CD25+ T cells isolated from mice infected with S. japonicum have played roles of Th1-dominant immune suppression.